PROJECT SUMMARY/ABSTRACT The OVERALL OBJECTIVE of this renewal is to clarify the pathogenesis of Primary Sclerosing Cholangitis (PSC) and develop effective treatments. PSC affects 30,000 Americans, accounts for 6% of liver transplants, and has a median survival of 13-20 years. Characterized by a fibroinflammatory peribiliary microenvironment, it progresses to cirrhosis, is associated with inflammatory bowel disease, and has an increased risk of colon, bile duct, and gallbladder cancers. There are no approved drugs for PSC because its pathogenesis is obscure. While cholangiocytes are important in pathogenesis, how they contribute to PSC initiation and progression, and what cellular processes and molecules are involved is unclear. We reported that: i) cellular senescence (cell cycle arrest) and the senescence associated secretory phenotype (SASP) are features of cholangiocytes in PSC patients and animal models; and ii) key regulatory mechanisms include a central molecular pathway (NRAS/MEK/ERK), upregulation of a transcription factor, ETS1, and epigenome modifications (loss of H3K27me3). These results suggest that ETS1 functions as an essential mediator of cholangiocyte senescence. We also found that: i) induction of cholangiocyte senescence in vitro causes hypersecretion of fibroinflammatory molecules (i.e., SASP) and apoptosis resistance from an imbalance of pro- and anti- apoptotic proteins. RNAseq on cholangiocytes from PSC patients showed upregulation of genes associated with SASP and apoptosis resistance and enrichment of ETS1 target sites within their promoters. By ChIP- qPCR, we found that ETS1 and the histone acetyltransferase, p300, interact and localize at senescence, prosurvival, and SASP associated loci. Finally: i) in vitro, pro-apoptotic, BH3-only mimetics, kill senescent but not non-senescent cholangiocytes; and ii) in vivo, killing senescent cholangiocytes using either novel, genetic cross-breeding (Mdr2-/--INK-ATTAC mouse) or pharmacologic treatment (BH3 mimetic) reduces peribiliary fibrosis. Thus, our data support the CENTRAL HYPOTHESIS that in PSC, ETS1 promotes chromatin remodeling and selective transcription inducing cholangiocyte senescence, apoptosis resistance, and SASP resulting in hypersecretion of bioactive molecules that induce a fibroinflammatory peribiliary microenvironment. We will test this hypothesis using novel in vitro biochemical and molecular techniques, and in vivo using animal models. We have 3 SPECIFIC AIMS. First, we test the hypothesis that ETS1 and p300 mediate cholangiocyte senescence. Second, we test the hypothesis that ETS1 mediates apoptosis resistance by upregulation of pro-survival proteins, including BCL- XL. Third, we test the hypothesis that ETS1 mediates cholangiocyte SASP promoting a fibroinflammatory peribiliary microenvironment through upregulation of CXCL12. Results from our innovative experiments will clarify the pathways and molecules involved in the development of PSC and could lead to novel therapies targeting a PSC cholangiocyte signature of senescence, apoptosis resistance, and SASP.